The aim of the proposed project is to investigate the mitochondrial protein degradation system - a largely neglected system involved in the biogenesis and maintenance of mitochondria and mitochondrial components. We will investigate the mitochondrial protein degrading system in yeast - the organism most frequently used in studies of mitochondrial biogenesis. ATP-dependent protease, a major component of the mitochondrial protein degrading machinery, will be for the first time isolated from yeast mitochondria. The enzyme will be characterized and compared with its mammalian counterpart and with bacterial protease La. Antibodies against the enzyme will be raised and used to determine the enzyme levels under the conditions affecting mitochondrial biogenesis. N-terminus of the enzyme will be microsequenced with the aim to clone the corresponding gene. Hydrolysis of normal and mutationally altered forms of yeast cytochrome c will be compared to the corresponding half-lives in vivo. Possible heat-shock character of the enzyme will be tested and its functional relationship with mitochondrial chaperonins will be examined. ATP-independent protease(s) of yeast mitochondria will be also purified and characterized. The level and activities of the yeast mitochondrial ATP-dependent protease will be examined in yeast under conditions that affect mitochondrial biogenesis, such as catabolite repression and anaerobic grow. A functional link between the mitochondrial respiratory enzymes and the ATP-dependent protease will be investigated. These results should shed light on the hypothesis that the mitochondrial degradation system regulates steady-state level of normal mitochondrial proteins, as well as eliminates abnormally unstable forms.